network mechanisms of theta related neuronal activity in hippocampal ca1 pyramidal neurons
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Spatio-temporally correlated neuronal activity is commonly observed as network oscillations or rhythmic activity in neuronal circuits1–5. One such rhythm is a relatively slow, 4–10-Hz oscillation of extracel-lular current that has been found in the hippocampal formation of all mammals studied to date1,4. This theta oscillation appears to provide a timing signal to hippocampal firing that is thought to be important for both spatial and nonspatial hippocampal functions during active exploration and REM sleep1–4,6–10. Three characteristic firing6,7,9–11 and subthreshold12 membrane potential features have been reported to occur in place cells as the animal moves into and through the neu-rons’ place field. First, firing rate increases are associated with steadily increasing levels of intracellular depolarization. Second, the time of peak intracellular depolarization and action potential output both advance relative to the extracellular theta phase. Finally, as the animal leaves the place field, the firing rate decreases back to baseline levels while the timing of the action potentials stays advanced. The available intracellular data suggest that the peak intracellular depolarization remains advanced for this portion.
Generally, two forms of fairly abstract models have been proposed to account for the place-related firing rates and phase precession. These include several forms of oscillatory interference models6,13–18 as well as a more systems-level model involving cell assemblies and asymmetric synaptic connections among the active neurons13,19,20. Most of the above models are expected to produce characteristic intra- and extracellular signals that markedly differ from those described above12. However, one particular interference model, the soma- dendrite interference (SDI) model, has been proposed to account for many of the appropriate features10,12,15,16. We sought to establish a physiological implementation of the SDI model using specific cel-lular- and network-level mechanisms to produce the characteristic features of theta-related neuronal activity.
RESULTSThe SDI model attempts to capture the flow of activity in the hippo-campal network during theta states and has two main components, a phasic excitatory input to the dendrites that is simultaneous with a similarly phasic inhibitory input to the perisomatic regions6,15–18. We used simultaneous whole-cell somatic and distal apical dendrite recordings in CA1 pyramidal neurons located in acute hippocampal slices from parvalbumin-Cre (PV-Cre) transgenic mice that virally express a channelrhodopsin2–green fluorescent protein fusion protein (ChR2-GFP; Supplementary Fig. 1 and Online Methods) in parvalbumin-expressing interneurons21,22 to compare the ability of two different versions of a SDI model to reproduce theta oscil-lation–associated hippocampal activity. In both versions, dendritic excitation was provided by a progressively increasing depolarizing current injection in the shape of a 5-Hz rectified sine wave into the distal apical trunk (Fig. 1). In the first form of implementation, the dendritic depolarization was paired with a 180° out of phase (that is, hyperpolarizing) somatic current sine wave injection of constant amplitude (100–150 pA)12–16. Current-voltage relationships from the soma and dendrite indicated that the form of inhibition produced by this SDI model was subtractive in that the slope of the relationship was only mildly altered (soma, 7 ± 1% Δgain/gain, n = 7; dendrite, 9 ± 5% Δgain/gain, n = 4), whereas a large rightward shift was produced (soma, 104 ± 2 pA, n = 7; dendrite, 59 ± 5 pA, n = 4; Fig. 1d,e).
In the second form of SDI, a divisive form of phasic somatic inhibi-tion was implemented. To achieve this, we used one-photon (Fig. 1a–c and Supplementary Fig. 1) or temporally focused two-photon (2pTeFo; see Online Methods and Supplementary Fig. 2)23 ChR2 photostimulation confined to the cell body layer of the hippocampal CA1 region with a temporal pattern that produced a rectified 5-Hz sine wave of inhibitory conductance. This spatio-temporal pattern of
1Department of Neuroscience, Columbia University, New York, New York, USA. 2Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia, USA. 3These authors contributed equally to this work. Correspondence should be addressed to J.C.M. ([email protected]).
Received 16 February; accepted 14 June; published online 18 July 2010; doi:10.1038/nn.2597
Network mechanisms of theta related neuronal activity in hippocampal CA1 pyramidal neuronsAttila Losonczy1,3, Boris V Zemelman2,3, Alipasha Vaziri2 & Jeffrey C Magee2
Although hippocampal theta oscillations represent a prime example of temporal coding in the mammalian brain, little is known about the specific biophysical mechanisms. Intracellular recordings support a particular abstract oscillatory interference model of hippocampal theta activity, the soma-dendrite interference model. To gain insight into the cellular and circuit level mechanisms of theta activity, we implemented a similar form of interference using the actual hippocampal network in mice in vitro. We found that pairing increasing levels of phasic dendritic excitation with phasic stimulation of perisomatic projecting inhibitory interneurons induced a somatic polarization and action potential timing profile that reproduced most common features. Alterations in the temporal profile of inhibition were required to fully capture all features. These data suggest that theta-related place cell activity is generated through an interaction between a phasic dendritic excitation and a phasic perisomatic shunting inhibition delivered by interneurons, a subset of which undergo activity-dependent presynaptic modulation.
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input produced a divisive form of inhibition that originated predomi-nantly around the soma. This was evidenced by a large decrease in the slope (soma, 48 ± 3% Δgain/gain, n = 7; dendrite, 19 ± 6% Δgain/gain, n = 4; Fig. 1d,e) and a relatively small rightward shift of the somatic current-voltage (soma, 19 ± 6 pA, n = 7; dendrite, 10 ± 4 pA, n = 4), as well as by the kinetics of the evoked unitary inhibitory responses (n = 8; Supplementary Fig. 1). Similar effects were observed on the suprathreshold firing frequency-input relationships in which the stimulation of perisomatic inhibition reduced the slope of the rela-tionship (Δgain/gain = 48%, shift = 170 pA, n = 7) to a much greater extent than the injection of hyperpolarizing currents (Δgain/gain = 13%, shift = 117 pA, n = 7; Supplementary Fig. 3). The rightward shift in the frequency-input relationship induced by an inhibitory synaptic conductance is likely a result of the relatively low levels of synaptic noise present in the inputs24,25. These relationships sug-gest that for near threshold levels of excitatory input (~250 pA), the dendritic arbor of CA1 pyramidal neurons would be relatively more depolarized in the presence of shunting perisomatic inhibitory input than with subtractive inhibitory input (shunt, 27 ± 2 mV; subtractive, 20 ± 2 mV; n = 4, P < 0.01), even though the somatic regions will be depolarized to similar levels (shunt, 9 ± 1 mV; subtractive, 11 ± 1 mV; n = 4, P > 0.05). Such elevated dendritic depolarization could increase the probability of dendritic plateau potential generation26.
We next implemented the SDI model using either a subtractive or divisive form of somatic inhibition and compared the intracellularly recorded profile of spike timing and subthreshold membrane poten-tial during progressively increased excitation. The subtractive inhibi-tion form of SDI produced a pattern of action potential output that
was characterized by a very controlled precession of spike times from ~270° to ~90° when referenced to the somatic hyperpolarizing sine wave (that is, external reference; Figs. 2 and 3, and Supplementary Fig. 4). The profile for the subthreshold potentials (3–8-Hz bandpass-filtered traces with action potentials removed where applicable, see Online Methods) was very similar in that the peak of the depolari-zation gradually moved forward in time with increasing excitation (Fig. 3a). Because the peak depolarization of the subthreshold poten-tial advanced in time nearly as much as action potential latencies, there was very little phase precession of the spikes when they were referenced to the intracellular somatic potential waveforms (that is, internal reference; Fig. 3a). Notably, the peak-to-trough amplitudes of the subthreshold potentials showed a biphasic relationship with excitation, as they initially decreased for several cycles before finally increasing in amplitude as excitation enhanced (Figs. 2 and 3, and Supplementary Fig. 4). Together, these data indicate that, in the sub-tractive form of the SDI model, an interaction of the filtered dendritic excitatory input and the unfiltered somatic hyperpolarization pro-duces an intrasomatic potential whose depolarizing peak smoothly advances in time and phase driving action potential initiation to shorter and shorter latencies. The amplitude of the subthreshold somatic potential, initially determined by the size of the large somatic hyperpolarization (~−10 mV), first decreased as the progressively increasing depolarization from the dendrite gradually removed the hyperpolarizing component and then increased as the depolarizing component dominated the subthreshold potential shape.
The divisive form of SDI produced a different profile. As with the subtractive form, increasing dendritic excitation induced a gradual
Figure 1 Experimental implementation of subtractive and divisive forms of inhibition. (a) Schematic of the experimental configuration. A dendritic wholecell electrode was used to inject a depolarizing sine wave current while the corresponding somatic and dendritic voltage responses were recorded in the absence or presence of a somatic 180° outofphase hyperpolarizing sine wave current injection or ChR2 photostimulation. (b) Twophoton image stack of a CA1 pyramidal neuron (red, Alexa 594) in a dense network of ChR2GFP–positive fibers (green) concentrated in the pyramidal layer in a hippocampal slice from a PVCre mouse injected with rAAVFLEXrevChR2GFP. Blue dots represent the ChR2 photostimulation grid. (c) Individual (left, 1–25 locations, 100ms interval) and synchronous (right, 1–25 locations, 3ms interval) inhibitory postsynaptic currents (VC, voltage clamp) potentials (CC, current clamp) evoked by photostimuli corresponding to the locations indicated by letters in b (a: 1st, 13rd and 25th; b: 2nd and 14th; c: 3rd and 15th; d: 4th and 16th; e: 5th and 17th; f: 6th and 18th; g: 7th and 19th; h: 8th and 20th; i: 9th and 21st; j: 10th and 22nd; k: 11th and 23rd; l: 12th and 24th points) and recorded at three different membrane potentials. Blue tickmarks represent timing and relative intensity of laser pulses. (d) Dendritic (upper, dend. Vm) and somatic (lower, soma Vm) membrane potential responses obtained for dendritic current injection of increasing amplitude alone (left), in the presence of somatic hyperpolarizing current injection (middle) or with ChR2 photostimulation (right). (e) Subthreshold inputoutput curves recorded at the dendrite (upper) and at the soma (lower) for the three different input conditions.
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phase precession of action potential output that covered a phase range of ~180° (~270° to ~90°) when referenced to the somatic hyperpolarizing current wave (external reference; Figs. 2 and 3, and Supplementary Figs. 2 and 4). Again, the depolarizing peak of the subthreshold somatic potential that drove the precession showed sim-ilar levels of phase advance (Fig. 3b) and therefore very little action potential phase precession could be observed when the spike laten-cies were referenced to the intracellular potential waveform (internal reference; Fig. 3b). However, with the inhibition being generated by a predominantly perisomatic conductance, the subthreshold poten-tial amplitude increased monotonically with the progressive increase in excitatory input (Figs. 2b and 3d, and Supplementary Fig. 4). The reason for this is that with the inhibitory reversal potential near the resting membrane potential there was very little hyperpolarizing
current generated at the lowest excitatory input levels, even though there is a large inhibitory conductance. Thus, a shunting or divisive form of inhibition produced enough perisomatic inhibition to allow both appropriate increases in firing rate and phase precession during periods of elevated dendritic excitation without also producing large membrane hyperpolarizations when excitation is weaker. This is a very similar profile to that recently recorded in behaving animals12.
Dendritic excitation alone, without any form of inhibition, pro-duced a subthreshold potential amplitude profile that, as described above, increased monotonically with increased excitation (Figs. 2c and 3c, and Supplementary Figs. 2 and 4). In contrast with the SDI models, however, the amount of phase precession observed for out-put action potentials or for the subthreshold potentials was mark-edly reduced to less than half of the range seen in either form of
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Figure 2 SDI induces phase precession. (a) Dendritic excitation plus somatic current injection. Concatenation of dendritic current injection (upper gray, 0, 40, 75, 130, 200, 300, 500, 670 and 1,300 pA), soma membrane potential (soma Vm, dark blue), 3–8Hz bandpassfiltered Vm (black) and soma current injection (light blue) waveforms. Hyperpolarizing 5Hz sine wave–shaped somatic current injections (100pA constant amplitude) were paired with depolarizing 5Hz sine wave–shaped current injections into the distal apical dendrites. As the dendritic current amplitude was increased, the spike latency (upper) and timing of the subthreshold peak depolarization (lower) advanced (gray dots, depolarization peak; black tickmarks, spike peak). (b) Dendritic excitation plus perisomatic conductance stimulation. Concatenation of dendritic current injection (upper gray, 0, 40, 75, 130, 200, 300, 500, 670 and 1,300 pA), soma Vm (dark green), bandpassfiltered Vm (black) and approximate perisomatic conductance (light green) waveforms. Increasing dendritic depolarizing current injection was paired with 5Hz sine wave–shaped ChR2 photostimulation. Spike latency (upper) and timing of the subthreshold peak depolarization (lower) advanced with increased dendritic current (gray dots, depolarization peak; black tickmarks, spike peak). (c) Dendritic excitation only. Concatenation of dendritic current injection (upper gray, 40, 75, 130, 200, 250, 300, 500, 670 pA), soma Vm (dark red), bandpassfiltered Vm (black) and missing soma inhibitory (light red dashed) waveforms. Increasing dendritic current injection was delivered alone without somatic inhibition. Spike latency (upper) and timing of the subthreshold peak depolarization (lower) advanced only slightly with increased dendritic current (gray dots, depolarization peak; tickmarks, spike peak).
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the SDI models (excitation Alone, 58 ± 8°, n = 6; with subtractive inhibition, 172 ± 14°, n = 7; with divisive inhibition, 146 ± 21°, n = 7; P < 0.01), regardless to which waveform the spike times were referenced (Fig. 3c). Thus, in the end, only the SDI model using a divisive form of shunting perisomatic inhibition was able to capture the first two of the characteristic features reported recently for hippocampal theta activity12: a steadily increasing level of somatic depolarization that accompanies the increase in action potential rate and the time of peak subthreshold depolarization advances nearly as much as the evoked action potentials such that action potentials are observed to phase advance only when referenced to the cycle of the somatic input. It is important to note that the presence of either form of perisomatic inhibition caused spike timing to be delayed relative to that produced by excitation alone. Thus, perisomatic inhibition produced phase precession by spreading out spike timing over an entire theta phase, with the exact timing being directly dependent on the level of phasic excitatory dendritic input.
A notable and poorly understood feature of theta-related place cell activity is that, as an animal leaves the place field, the phase and rate of place cell firing become dissociated while the phase of firing stays advanced and the firing rate declines27–29. Neither of the above implementations of the SDI model would be expected to capture this feature, as the phase of firing and peak subthreshold depolarization simply shift back in time as the excitation level and firing rate decrease back to baseline levels. How could the SDI model be modified to pro-duce a monotonic phase advance in the face of a biphasic firing rate
profile? We reasoned that the temporal profile of perisomatic inhibi-tion might become altered after a few theta cycles of high-frequency action potential firing as a result of activity-dependent modulation of GABA release from inhibitory terminals. To test this, we changed the symmetry of inhibition by relatively delaying (Fig. 4a,b) the peak of inhibition, as would be expected for a shift from short-term synaptic depression to facilitation. Indeed, we found that, when decreasing levels of phasic dendritic excitation were paired with a phasic peri-somatic inhibition whose peak was now relatively delayed in time (Δpeak ginh, 63 ± 5°, n = 9), the firing rate gradually declined while the peak phase of subthreshold polarization and the firing phase stayed constantly precessed (Fig. 4c–e). The reason for this is that inhibition with a delayed peak conductance effectively shunted out the peak of the filtered dendritic depolarization at the soma and blocked spiking later in the cycle even for high levels of excitation (Supplementary Fig. 5). In contrast, phasic inhibition with peak relatively advanced in time (Δpeak ginh, 45 ± 4°, n = 4) moderately reduced the range of phase modulation (Supplementary Fig. 5) by confining spiking to later in the cycle. Together, we were able to successfully separate the firing rate from the firing phase and timing of peak subthreshold depolarization by delaying the peak of the perisomatic inhibition dur-ing periods of waning excitation.
What mechanism could underlie such an activity-dependent and locally mediated shift in the timing of perisomatic inhibition? It is well established that GABA release from inhibitory terminals is modu-lated by a wide repertoire of presynaptic auto- and heteroreceptors.
Figure 4 Delayed inhibition dissociates placecell firing rate and phase. (a) Left, experimental configuration: somatic filtered sinewave current injection paired with 2pTeFo ChR2 photostimulation of inhibitory profiles. Right, twophoton stack of a CA1 pyramidal neuron (red) and ChR2GFP–positive profiles of parvalbuminexpressing interneurons. Grey dots indicate the photostimulation grid. Bottom right, individual inhibitory postsynaptic currents evoked by random pattern photostimulation. Red tickmarks represent 880nm laserpulse timing. (b) Stimulation sequence arranged to evoke a bimodal (upper left, green) or an increasing (lower left, blue) amplitude sequence that generated either a symmetrical (upper right, ginh (S); gray, individual traces; green, average) or a delayed (lower right, ginh (D); gray, individual traces; blue, average) inhibition profile with synchronous photostimulation (3ms interval), respectively. (c) Repeated 2pTeFo photostimulation: 1st–9th theta cycles with rampup depolarizing current injections (Idepol, dark gray) and symmetrical inhibition (Vm, soma ginh only, green), 10–18th theta cycles with rampdown current injections and delayed inhibition (Vm, soma ginh only, blue). Vm soma, soma membrane potential; filtered, 3–8Hz bandpassfiltered Vm; external reference, population inhibitory waveform. Black tickmarks, spike timing; gray dots, subthreshold peak. (d) Summary of mean spike phase (green and blue circles) and the mean phase of peak subthreshold potential (gray circles, mean ± s.e.m., n = 9) for increased and decreased levels of depolarization paired with symmetrical or delayed inhibition, respectively (n = 9). Solid lines, exponential fits. (e) Summary of mean number of spikes per cycle versus levels of current injections (mean ± s.e.m., n = 9). Solid lines, linear fits.
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We tested the potential role of type I cannabinoid receptors (CB1Rs), which are present in the axon terminals of cholecystokinin (CCK)-expressing interneurons30–32. To recruit axon terminals of CCK-expressing interneurons and to avoid direct depolarization of perisomatic GABAergic boutons on the recorded pyramidal neurons, we used high–spatial resolution 2pTeFo photostimulation localized to the CA1 cell body layer in slices from GAD65-Cre knock-in mice that virally expressed ChR2-GFP in GABAergic hippocampal inter-neurons. We found that repetitive 2pTeFo photostimulation23 of CB1R agonist–sensitive inhibitory fibers (Fig. 5) evoked an inhibitory con-ductance with peak phase that was shifted to later in the cycle (peak ginh, 127 ± 27°, n = 5) as a result of the short-term synaptic facilitation present at these terminals in the presence of the CB1R agonist (1 μM WIN55,212-2). Subsequent application of CB1Rs antagonist AM251 (10 μM) reversed the short-term facilitation to short-term synaptic depression and this temporally advanced the peak of the inhibition (peak ginh, 53 ± 10°, n = 5, P < 0.05) on pyramidal cells from GAD65-Cre mice. In contrast, the short-term plasticity of GABA release and the temporal profile of photostimulation-evoked inhibitory responses were insensitive to pharmacological manipulations of CB1Rs in the PV-Cre mice (n = 2; Fig. 5c). These data suggest that an activity-dependent release of endocannabinoids from active place cells during later theta cycles may contribute to the pattern of theta related activity through the local modulation of the temporal profile of perisomatic GABA release33,34. Together, our results indicate that a shift in the relative timing of perisomatic inhibition paired with the waning of dendritic excitation can produce the dissociation of place cell firing rate and phase observed as an animal leaves the center of the field.
DISCUSSIONOur data provide new biophysical mechanisms for hippocampal theta-related neuronal activity by demonstrating that many of the reported
features of both intra- and extracellularly recorded theta activity are captured by a SDI model implemented by a divisive or shunting form of perisomatic inhibition, whose temporal profile is modulated in an activity-dependent manner (Supplementary Fig. 6). Specifically, our results support the following framework for the generation of hippo-campal theta-related neuronal activity. As the animal moves into a place field of a particular CA1 pyramidal neuron, the increase in excitatory drive to the neuron, via the CA3 Schaffer collateral inputs, induces progressively more action potential firing (higher rate) with the timing of these spikes shifting forward with respect to the basket cell timing signal because of an interference between the dendritic and somatic oscillations. Furthermore, the prolonged activity of the place cell will result in the retrograde release of endocannabinoids33,34. As the animal begins to leave the place field, decreasing levels of excita-tion, together with an endocannabinoid-mediated local modulation of the temporal profile of perisomatic inhibition, perhaps further aided by increased levels of asynchronous release from CCK-expressing terminals35,36, result in a decrease in the action-potential firing rate while the timing of the spikes stays advanced. This proposed role of activity-dependent presynaptic modulation of GABA release is sup-ported by recent reports of profound alteration in the coordination and phase precession of place-cell spiking activity by cannabinoids in vivo37,38. Our results further suggest that trial-to-trial fluctuations in the magnitude of presynaptic modulation of GABA release, perhaps related to the running speed of the animal, might also contribute to the increase in spike phase variance in the last third of the place field for pooled-trial recordings observed in vivo39.
From a neural computation point of view, it appears that CA1 pyramidal neurons, as well as many others, may function as a comparator for multiple classes of inputs26,40,41. In the case of CA1 pyramidal neurons, an internal hippocampal representation provided by the CA3 Schaffer collateral input to the more proximal dendritic regions is compared with an external representation provided by the entorhinal cortical input to the dendritic tuft. When these inputs arrive with the proper timing in the same neuron, dendritic plateau potential initiation, burst-firing action potential output and robust synaptic plasticity are produced26. Our implementation of the SDI model using the highly compartmentalized form of shunting inhibi-tion produced phase precession that advanced action potential out-put without altering the integrative properties of the dendritic arbor. Given the requirement of backpropagating action potentials in these pathway interactions, the advance of up to 100 ms is an important feature of phase precession, as it greatly enhances the temporal over-lap of entorhinal cortical input and Schaffer collateral–driven CA1 output42. The combination of these two elements, phase precession without dendritic shunting or hyperpolarization, allows only those neurons receiving the proper input levels to output bursts of action potentials and robustly potentiate the involved synapses26. This potential ‘match signal’ resulting from a positive input comparison may be involved in hippocampal novelty detection and sequence retrieval43,44. Furthermore, as subcellular domain-specific innerva-tion of the neuronal surface by co-aligned sets of GABAergic and glutamatergic inputs is a general organizing principle in cortical circuits45, our results suggest that spatio-temporal interactions between excitatory and inhibitory inputs in and across different sub-cellular compartments may provide a general framework for cortical temporal coding.
The recent availability of accurate intracellular in vivo data has greatly aided the attempt to provide actual cellular and network mechanisms for behaviorally related neuronal activity12. In turn, our in vitro results produce several specific hypotheses that could be
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Figure 5 Presynaptic cannabinoid receptor activations delays the time to peak of inhibition. (a) CB1R agonist–sensitive inhibitory postsynaptic currents evoked by repeated 2pTeFo ChR2 photostimulation at a single location in the pyramidal layer in a GAD65Cre mice in the presence of CB1R agonist (1 μM WIN55,2122, blue) and following CB1R antagonist (10 μM AM251, green) application. Red tickmarks indicate the timing of the 2pTeFo photostimulation pulses at 880 nm. (b) Representative membrane potential traces of CB1R agonist–sensitive inhibitory responses evoked by burst 2pTeFo photostimulation (36 stimuli, one to six nonoverlapping locations repeated six times with a 1ms duration and a 3ms interval, ~40Hz cycle) alone (lower traces) and paired with depolarizing current injections (+200 pA, upper traces) in the presence of WIN55,2122 (1 μM, blue) and following subsequent application of AM251 (10 μM, green). Note that the timing of action potentials was shifted forward in the presence of CB1R agonist. Red tickmarks indicate the timing and relative intensity of the 2pTeFo photostimulation pulses at 880 nm. (c) Summary data of peak phase of inhibitory responses evoked by 2pTeFo photostimulation in PVCre mice (open circles, n = 2) and peak phase of CB1R agonist–sensitive inhibitory responses evoked by 2pTeFo photostimulation in GAD65Cre mice (right, solid gray circles, n = 5; black, mean ± s.e.m.) in the presence of WIN55,2122 (1 μM) and following subsequent application of AM251 (10 μM).
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tested with future in vivo recordings. First, silencing of CA1 basket cells (particularly parvalbumin-expressing) should remove phase precession by maximally advancing spike phase regardless of the location in the place field. In addition, silencing studies may be able to determine if the overall levels of perisomatic inhibition are con-stant across the place field, as suggested by the current SDI model. Future studies combining high-quality in vivo and in vitro recordings and manipulations in rodents will continue to advance our under-standing of the link between neuronal circuit computations and specific behaviors46.
METhODSMethods and any associated references are available in the online version of the paper at http://www.nature.com/natureneuroscience/.
Note: Supplementary information is available on the Nature Neuroscience website.
AcknowledgmentSWe thank B. Shield, D. O’Connor and A. Villacis for help with histology and stereotaxic viral injections, and B.K. Andrasfalvy for help with the temporal focusing experiments. We thank for P. Somogyi and S. Siegelbaum for their comments on a previous version of the manuscript. Precursors of the GAD65-Cre knock-in mice were originally developed by B.V.Z. in the laboratory of G. Miesenboeck at the Memorial Sloan-Kettering Cancer Center in New York.
AUtHoR contRIBUtIonSA.L. and J.C.M. performed electrophysiological experiments, analyzed the data and wrote the paper. B.V.Z. prepared plasmids, designed Cre recombinase–dependent rAAV-FLEX-rev-ChR2-GFP viruses, generated the GAD65-Cre knock-in mouse line and helped with the manuscript. A.V. designed and built the experimental setup for temporal focusing.
comPetIng FInAncIAl InteReStSThe authors declare no competing financial interests.
Published online at http://www.nature.com/natureneuroscience/. Reprints and permissions information is available online at http://www.nature.com/reprintsandpermissions/.
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ONLINE METhODSAll experiments were conducted in accordance with US National Institutes of Health guidelines and with the approval of the Janelia Farm Institutional Animal Care and Use Committee.
Virus and knock-in mouse preparation. The ChR2-GFP fusion protein sequence was cloned into an adeno-associated viral cassette containing the mouse synapsin promoter, a woodchuck post-transcriptional regulatory element (WPRE), SV40 poly-adenylation sequence and two inverted terminal repeats. We used codon-optimized ChR2-GFP encoding the amino acid substitution H134R. rAAV-FLEX-rev-ChR2-GFP21 was assembled using a modified helper-free system (Stratagene) as a serotype 2/7 (rep/cap genes) adeno-associated virus, and harvested and purified over sequential CsCl gradients as described previously47. PV-Cre knock-in mice have been described previously22. GAD65-Cre knock-in mice were generated using an IRES targeting construct to insert the Cre recom-binase coding sequence into the 3′ UTR of the mouse Gad65 (Gad2) gene. The construct was electroporated into a hybrid C57BL/6-129/SV stem cell line. F1 progeny were backcrossed repeatedly to C57BL/6 to recreate the C57BL/6 genetic background. C57BL/6 content of the resulting mice was confirmed by microsatellite testing (Charles River Laboratory).
Virus injection. PV-Cre and GAD65-Cre knock-in mice (3–5 weeks old) were anesthetized with isoflurane and placed in a stereotaxic apparatus (David Kopf Instruments). A small hole was drilled in the skull using a dental drill. Virus was delivered with a glass micropipette (tip size: 10 μm) using a microinjector (Nanoject II, Drummond Scientific). Dorsal hippocampus was injected at three sets of coordinates: 2.2, 2.4 and 2.7 mm posterior from bregma, and 2.1 mm from midline. We injected 20–30 nl of virus every 150 μm from 1.55 mm to 0.95 mm below pia. The pipette was held at 0.95 mm for 3 min before being completely retracted from the brain.
Hippocampal slice preparation and electrophysiology. PV-Cre and GAD65-Cre mice were infected with Cre-dependent rAAV-FLEx-rev-ChR2-GFP as described above. The mice were deeply anesthetized and decapitated 3–5 weeks post-injection. Coronal slices (350 μm) were prepared from the dorsal hippo-campus48 and visualized using an Olympus BX-61 epifluorescence microscope equipped with Dodt Gradient Contrast (Luigs and Neumann) under infrared illumination using a water-immersion lens (60× with 0.8 NA, Olympus) or with a Zeiss Examiner Z1 epifluorescence microscope equipped with Dodt Gradient and with a 20× objective (W-Plan-Apochromat, 1.0 NA, Zeiss). Experiments were performed at 32–35 °C in artificial cerebrospinal fluid containing 125 mM NaCl, 2.5 mM KCl, 25 mM NaHCO3, 1.25 mM NaH2PO4, 2 mM CaCl2, 1 mM MgCl2 and 25 mM glucose, and saturated with 95% O2 and 5% CO2. Simultaneous dual whole-cell patch-clamp recordings from distal dendrites and somata of hippo-campal CA1 pyramidal neurons (Figs. 1–3 and Supplementary Figs. 1, 3 and 4) or somatic whole-cell patch-clamp recordings (Figs. 4 and 5, and Supplementary Figs. 2, 4 and 5) were performed using Dagan BVC-700 amplifiers (Dagan) in the active bridge mode for voltage-clamp experiments, filtered at 1–3 kHz and digitized at 50 kHz. The distance of the dendritic electrode (mean ± s.e.m., 176 ± 8 mm, n = 14 recordings) was measured on stacked two-photon images collected at the end of recordings (ImageJ, US National Institutes of Health). Series resist-ance for somatic recordings was 10–25 MΩ, whereas that for dendritic recordings was 20–50 MΩ.
Recording electrodes were filled with an internal solution containing 120 mM potassium gluconate, 6 mM KCl, 10 mM HEPES, 4 mM NaCl, 8 mM Mg2ATP, 0.6 mM Tris2GTP, 28 mM phosphocreatine and 0.1 mM Alexa 594 (Molecular Probes), pH = 7.25, and the membrane potential was kept close to the GABA-A reversal potential measured in voltage clamp. Voltages were not corrected for the theoretical liquid junction potential (~8 mV).
To isolate GABA-A receptor–mediated responses, we prepared a GABA-B receptor antagonist (CGP55845A, Tocris) in DMSO as 1 mM stock solution and used it at 1 μM final concentration in most of the experiments. Under these condi-tions, GABA-A receptor antagonist SR95531 (20 μM, Tocris) completely blocked the photostimulation evoked responses on pyramidal cells (n = 4; Supplementary Fig. 1). CB1R agonist WIN 55,212-2–mesylate and CB1R antagonist AM251 (both from Tocris) were prepared in DMSO as 1 mM and 10 mM stock solutions and used at 1 μM and 10 μM final concentration, respectively.
one-photon and 2pteFo chR2 photostimulation. A dual galvanometer-based scanning system (Prairie Technologies, Middleton, WI) was used for 2pTeFo or one-photon ChR2 photostimulation and combined with two-photon imaging. For two-photon imaging, a 140-fs ultra-fast, pulsed laser beam at 920-nm center wavelength (Chameleon Ultra II, Coherent), controlled with an electro-optical modulator (Model 350-50, Conoptics), was used to excite Alexa 594 and GFP.
For one-photon laser ChR2 photostimulation (Figs. 1–3 and Supplementary Figs. 1, 4 and 5), a 473-nm blue laser (DPSS, CrystaLaser) was coupled to the uncaging path of the scan-head. Photostimuli consisted of light pulses of 1-ms duration and power levels in the range of 10–100 μW at the specimen. The timing, position and intensity of the light pulses were controlled using the lasers analog modulation circuitry in combination with a fast shutter (Uniblitz, LS2ZM2, Vincent Associates) via software written in LabView (PrairieView-TriggerSync, Prairie Technologies).
Experimental setup for two-photon temporal focusing was implemented as described previously23,49. Briefly, a 140-fs ultra-fast pulsed laser beam at 880-nm center wavelength (Chameleon Ultra II, Coherent) controlled with an elec-tro-optical modulator (Model 350-50, Conoptics) was used as the source for temporal focusing. To be able to move the temporally focused spot in a 100-μs timescale, we integrated it into the scan-head of a two-photon scanning upright microscope (Examiner Z1, Zeiss and Prairie Technologies). The beam was first reflected toward a ruled diffraction grating with 300 lines per mm (Richardson) from which the first order diffraction was used. The illuminated spot on the grating was then imaged onto the space between X and Y scanning mirrors using an achromatic planoconvex lens (Thorlabs, f = 1,000 mm). The scanning mir-rors scanned the temporally focused beam on the back focal aperture of a 20× objective (W-Plan-Apochromat, 1.0 NA, Zeiss). The temporal focusing light was reflected and combined with imaging light by a dichroic mirror (900dcxxr-laser, Chroma). The timing, position and intensity of the light pulses were controlled via software written in LabView (PrairieView-TriggerSync, Prairie Technologies). Temporal-focusing ChR2 photostimulation consisted of light pulses of 0.1– 1-ms duration and power levels in the range of 50–500 mW at the specimen. At nonsaturated power levels (<50 mW), temporal focusing yielded two-photon excitation of a disc-shaped volume with a diameter of ~5 μm and thickness of ~2 μm23. These values were measured using a thin (~100 nm) layer of fluores-cence material on a microscope cover glass that was axially scanned through the temporally focused excitation volume. However, mainly because of the out-of-focus excitation of ChR2 by the 2pTefo, the spatial resolution of excitation (axially and laterally) was slightly less confined when measured electrophysiologically using saturating power levels (~50–500 mW)23. We found that using such a defo-cused excitation volume (diameter, ~20 μm; thickness, ~40 μm) increased the probability of suprathreshold bouton stimulation while still substantially reducing the overlap and ChR2 desensitization when multiple closely placed stimulus loca-tions were used in the cell body layer. The improved spatiotemporal resolution of ChR2-photostimulation by 2pTeFo allowed us to precisely manipulate the temporal profile of inhibition (Fig. 4), stimulate ChR2-expressing perisomatic inhibitory profiles at theta frequency repetitively (Fig. 4 and Supplementary Fig. 2) and stimulate GABAergic (both parvalbumin-expressing and CCK-expressing) profiles in the pyramidal layer in GAD65-Cre mice without directly depolarizing inhibitory terminals on the recorded pyramidal cell (Fig. 5).
In experiments with simultaneous dual whole-cell recordings, when a rectified 5-Hz sinusoidal depolarizing current was injected to the dendrite and paired with one-photon ChR2 photostimulation–evoked perisomatic inhibition (Figs. 1–3 and Supplementary Figs. 1 and 4), dendritic sinusoidal currents with different amplitudes (50–1,000 pA) were injected during subsequent trials, interleaved with 30–60-s pauses to allow for ChR2 recovery from desensitization. In a subset of experiments to examine the effect of advanced and delayed inhibitory inputs on the range of phase modulation (Supplementary Figs. 4 and 5), and in those using 2pTeFo repeated photostimulation (Figs. 4 and 5 and Supplementary Fig. 2), we used a single somatic electrode to inject a filtered depolarizing sinusoidal current (25–670 pA). The shape of the filtered rectified somatic current injection was set to reproduce the somatic depolarization profiles obtained with dual soma-dendrite recordings (Supplementary Fig. 5).
To evoke CB1R agonist–sensitive IPSCs on CA1 pyramidal cells from GAD65-Cre mouse (Fig. 5), we selected six non-overlapping locations in the pyramidal layer (>20 μm from the recorded neuron) where the amplitude of 2pTeFo-evoked unitary IPSCs showed >20% reduction on CB1R agonist application (data not
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shown). These CB1R agonist–sensitive IPSCs were subsequently used during high-frequency 2pTeFo photostimulation (36 stimuli from six consecutive loca-tions repeated six times with a 1-ms duration and a 3-ms interval).
data analysis. To produce the subthreshold potentials shown in Figures 2 and 4, and in Supplementary Figures 2 and 4–6, we ran raw somatic voltage traces through a custom algorithm written in Igor Pro 6. Action potentials were first removed by blanking the traces from 2 ms before the action potential peak to 5 ms after the peak; the traces were subsequently linearly interpolated and bandpass filtered using a linear-phase finite impulse response filter with a 1-ms Hamming window between 3–8 Hz. To produce the traces shown in Figure 2 and in Supplementary Figure 5, we concatenated eight or nine individual traces, each for a different dendritic excitation level, together and then ran the entire sequence of traces through the filter algorithm. Because the 5-Hz excitatory den-dritic waves have the same time course as the somatic current or conductance waves, we used either of these as a reference for the extracellular reference for the three experimental conditions. The phase of a spike occurring at time t was cal-culated as 360 × (t/200 ms). When referenced to the intracellular depolarization,
we calculated the spike phase as 360 × (t/tp), where tp was the time of the peak of the filtered intracellular potential. Changes in gain for the current-voltage and frequency-input curves were calculated as (control slope – experimental slope)/control slope, where the slope was determined from linear fits to the data. For frequency-input curves, input shifts were determined from the xhalf
of sigmoid functions base halfrate
+
+−
max
1 ex x , where base is the baseline frequency, max is
maximum frequency and rate is the slope parameter. The significance of differ-ences was tested with Student’s t test or ANOVA. P < 0.05 was considered to be significant. Data are given in mean ± s.e.m.
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